Use of biologically active vitamin D compounds for the prevention and treatment of inflammatory bowel disease

ABSTRACT

Methods of treating inflammatory bowel disease are described, and in particular the prevention and treatment of inflammatory bowel disease in humans as well as other animals. These methods involve the administration of biologically active vitamin D compounds, and therapeutic compositions thereof, so that the symptoms of Inflammatory Bowel Disease are reduced or relieved.

The present Application is a Continuation of application Ser. No.09/469,985, filed Dec. 21, 1999, which is now U.S. Pat. No. 6,358,939.

FIELD OF THE INVENTION

The present invention relates to therapeutics for the prevention andtreatment of inflammatory bowel disease, and in particular theprevention and treatment of inflammatory bowel disease in humans as wellas other animals through the use of biologically active vitamin Dcompounds.

BACKGROUND OF THE INVENTION

Inflammatory bowel diseases (IBD) are defined by chronic, relapsingintestinal inflammation of obscure origin. IBD refers to two distinctdisorders, Crohn's disease and ulcerative colitis (UC). Both diseasesappear to involve either a dysregulated immune response to GI tractantigens, a mucosal barrier breach, and/or an adverse inflammatoryreaction to a persistent intestinal infection. The GI tract luminalcontents and bacteria constantly stimulate the mucosal immune system,and a delicate balance of proinflammatory and anti-inflammatory cellsand molecules maintains the integrity of the GI tract, without elicitingsevere and damaging inflammation [MacDermott, R. P., J.Gastroenterology, 31:907:-916 (1996)]. It is unknown how the IBDinflammatory cascade begins, but constant GI antigen-dependentstimulation of the mucosal and systemic immune systems perpetuates theinflammatory cascade and drives lesion formation.

There is no known cure for IBD, which afflicts 2 million Americans.Current methods of managing IBD symptoms cost an estimated $1.2 billionannually in the United States alone.

In patients with IBD, ulcers and inflammation of the inner lining of theintestines lead to symptoms of abdominal pain, diarrhea, and rectalbleeding. Ulcerative colitis occurs in the large intestine, while inCrohn's, the disease can involve the entire GI tract as well as thesmall and large intestines. For most patients, IBD is a chroniccondition with symptoms lasting for months to years. It is most commonin young adults, but can occur at any age. It is found worldwide, but ismost common in industrialized countries such as the United States,England, and northern Europe. It is especially common in people ofJewish descent and has racial differences in incidence as well. Theclinical symptoms of IBD are intermittent rectal bleeding, crampyabdominal pain, weight loss and diarrhea. Diagnosis of IBD is based onthe clinical symptoms, the use of a barium enema, but directvisualization (sigmoidoscopy or colonoscopy) is the most accurate test.Protracted IBD is a risk factor for colon cancer. The risk for cancerbegins to rise significantly after eight to ten years of IBD.

Some patients with UC only have disease in the rectum (proctitis).Others with UC have disease limited to the rectum and the adjacent leftcolon (proctosigmoiditis). Yet others have UC of the entire colon(universal IBD). Symptoms of UC are generally more severe with moreextensive disease (larger portion of the colon involved with disease).

The prognosis for patients with disease limited to the rectum(proctitis) or UC limited to the end of the left colon(proctosigmoiditis) is better then that of full colon UC. Brief periodictreatments using oral medications or enemas may be sufficient. In thosewith more extensive disease, blood loss from the inflamed intestines canlead to anemia, and may require treatment with iron supplements or evenblood transfusions. Rarely, the colon can acutely dilate to a large sizewhen the inflammation becomes very severe. This condition is calledtoxic megacolon. Patients with toxic megacolon are extremely ill withfever, abdominal pain and distention, dehydration, and malnutrition.Unless the patient improves rapidly with medication, surgery is usuallynecessary to prevent colon rupture.

Crohn's disease can occur in all regions of the gastrointestinal tract.With this disease intestinal obstruction due to inflammation andfibrosis occurs in a large number of patients. Granulomas and fistulaformation are frequent complications of Crohn's disease. Diseaseprogression consequences include intravenous feeding, surgery andcolostomy.

The most commonly used medications to treat IBD are anti-inflammatorydrugs such as the salicylates. The salicylate preparations have beeneffective in treating mild to moderate disease. They can also decreasethe frequency of disease flares when the medications are taken on aprolonged basis. Examples of salicylates include sulfasalazine,olsalazine, and mesalamine. All of these medications are given orally inhigh doses for maximal therapeutic benefit. These medicines are notwithout side effects. Azulfidine can cause upset stomach when taken inhigh doses, and rare cases of mild kidney inflammation have beenreported with some salicylate preparations.

Corticosteroids are more potent and faster-acting than salicylates inthe treatment of IBD, but potentially serious side effects limit the useof corticosteroids to patients with more severe disease. Side effects ofcorticosteroids usually occur with long term use. They include thinningof the bone and skin, infections, diabetes, muscle wasting, rounding offaces, psychiatric disturbances, and, on rare occasions, destruction ofhip joints.

In IBD patients that do not respond to salicylates or corticosteroids,medications that suppress the immune system are used. Examples ofimmunosuppressants include azathioprine and 6-mercaptopurine.Immunosuppressants used in this situation help to control IBD and allowgradual reduction or elimination of corticosteroids. However,immunosuppressants cause increased risk of infection, renalinsufficiency, and the need for hospitalization.

Clearly there is a great need for agents capable of preventing andtreating IBD. It would be desirable if such agents could be administeredin a cost-effective and timely fashion, with a minimum of adverse sideeffects.

Definitions

The phrase “vitamin D compounds” include, but are not limited tocompounds which have at least one of the following features: the C-ring,D-ring and 3β-hydroxycyclohexane A-ring of vitamin D interconnected bythe 5,7 diene double bond system of vitamin D together with any sidechain attached to the D-ring (i.e. compounds with a ‘vitamin D nucleus’and substituted or unsubstituted A-, C-, and D-rings interconnected by a5,7 diene double bond system typical of vitamin D together with a sidechain attached to the D-ring).

The phrase “nonsecosteroidal vitamin D mimics” is defined asnonsecosteroid compounds which are capable of mimicking variousactivities of the secosteroid calcitriol. Examples of such compoundsinclude, but are not limited to, LG190090, LG190119, LG190155, LG190176,and LG1900178 [See, Boehm et al., Chemistry & Biology 6:265-275 (1999)].

The phrase “biologically active vitamin D compound” is defined asencompassing vitamin D compounds and nonsecosteroidal vitamin D mimicswhich are biologically active in vivo, or are acted upon in a subject(i.e. host) such that the compound becomes active in vivo. Examples ofsuch compounds include, but are not limited to: vitamin D, 1,25dihydroxyvitamin D₃ (1,25(OH)₂D₃) [a.k.a. calcitriol], and analogsthereof [e.g. 1α-hydroxyvitamin D₃ (1α-OH-D₃), 1,25-dihydroxyvitamin D₂(1,25-(OH)₂D₂), 1α-hydroxyvitamin D₂ (1α-OH-D₂), 1α,25-(OH)₂-16-ene-D₃,1α,25-(OH)₂-24-oxo-16-ene-D₃, 1α,24R(OH)₂-D₃, 1α,25(OH)₂₋₂₂-oxa-D₃,20-epi-22-oxa-24a,24b,-dihomo-1α,25(OH)₂-D_(3, 20)-epi-22-oxa-24a,26a,27a,-trihomo-1α,25(OH)₂-D₃,20-epi-22-oxa-24homo-1α,25(OH)₂-D₃,1,25-(OH)₂-16,23E-diene-26-trifluoro-19-nor-D₃, and nonsecosteroidalvitamin D mimics. Further examples are provided below, including variousstructural formulas, detailed in part III.

The phrase “symptoms of IBD” is herein defined to include symptoms,including, but not limited to, abdominal pain, diarrhea, rectalbleeding, weight loss, fever, loss of appetite, and other more seriouscomplications, such as dehydration, anemia and malnutrition. A number ofsuch symptoms are subject to quantitative analysis (e.g. weight loss,fever, anemia, etc.). Some symptoms are readily determined from a bloodtest (e.g. anemia) or a test that detects the presence of blood (e.g.rectal bleeding).

The phrase “calcemic response” is herein defined as the biologicalresponse caused by many biologically active vitamin D compounds (e.g.calcitriol) when administered to a subject. The response includes, butis not limited to, elevated calcium concentrations in serum, increasedintestinal absorption of dietary calcium, increased urinary calciumexcretion, and increased bone calcium mobilization.

The phrase “symptoms of hypercalcemia” is herein defined to detectedsymptoms including, but not limited to, calcium deposition in thekidneys (nephrocalcinosis), kidney stones (nephrolithiasis), uremia,manifestations of muscle weakness, lethargy, coma, constipation,anorexia, nausea, vomiting, shortening of the QT interval, hypotension,and arrhythmias.

The phrase “serious hypercalcemia” is herein defined as the conditionwhere a subject is suffering from symptoms of hypercalcemia whichrequire immediate medical attention to prevent life threatening illnessor death. Examples include, but are not limited to, nephrocalcinosis,nephrolithiasis, uremia, coma, and anorexia.

The phrase “mild hypercalcemia” is herein defined as the condition wherea subject is suffering from symptoms of hypercalcemia which do notrequire immediate medical attention to prevent life threatening illnessor death. Examples include, but are not limited to, manifestations ofmuscle weakness, lethargy, constipation, nausea, vomiting, shortening ofthe QT interval, hypotension, and arrhythmia.

The phrase “a therapeutically effective amount” of a biologically activevitamin D compound is herein defined as the dosage level required for apatient such that the symptoms of IBD are reduced.

The phrase “under conditions such that the symptoms are reduced” refersto any degree of qualitative or quantitative reduction in detectablesymptoms of IBD, including but not limited to, a detectable impact onthe rate of recovery from disease (e.g. rate of weight gain), or thereduction of at least one of the following symptoms: abdominal pain,diarrhea, rectal bleeding, weight loss, fever, loss of appetite,dehydration, anemia, distention, fibrosis, inflamed intestines andmalnutrition.

The phrase “at risk for IBD” is herein defined as encompassing thesegment of the world population that has an increased risk (i.e. overthe average person) for IBD. IBD is most commonly found in young adults,but can occur at any age. It occurs worldwide, but is most common in theUnited States, England, and northern Europe. It is especially common inpeople of Jewish descent. An increased frequency of this condition hasbeen recently observed in developing nations. Increased risk is alsopresent in people with family members who suffer from inflammatory boweldisease.

The phrase “therapeutic composition comprising biologically activevitamin D compounds” refers to compositions containing the biologicallyactive vitamin D compounds of the present invention, or the biologicallyactive vitamin D compounds of the present invention provided togetherwith one or more other compounds or agents including, but not limitedto, other biologically active vitamin D compounds, physiologicallytolerable liquids, gels, carriers, diluents, adjuvants, excipients,salicylates, steroids, immunosuppressants, antibodies, cytokines,antibiotics, binders, fillers, preservatives, stabilizing agents,emulsifiers, and buffers.

The phrase “continuous manner” when used in reference to the method ofdelivery or administration of the biologically active vitamin Dcompounds of the present invention, is defined as meaning asubstantially uninterrupted administration of the compounds of thepresent invention, such that a therapeutic dosage is stretched over aperiod of time and avoids a dosage ‘spike’ which is common among othermodes of administration (e.g. oral administration or intravenousadministration). Examples of modes of administration which employ acontinuous manner of delivery include, but are not limited to, atransdermal patch, a suppository, or a slow release oral formulation.

The word “subject” refers to a patient which is administered thetherapeutic composition comprising biologically active vitamin Dcompounds of the present invention. Examples of subjects, include, butare not limited to, humans and other animals such as non-human primates,horses, dogs, and cats.

SUMMARY OF THE INVENTION

The present invention relates to therapeutics for the prevention andtreatment of IBD. Specifically, the present invention contemplates theprevention and treatment of IBD in humans as well as other animalsthrough the use of biologically active vitamin D compounds.

The present invention provides a method of treatment, comprising;providing a subject and a therapeutic composition comprising abiologically active vitamin D compound; and administering thetherapeutic composition to the subject. It is not intended that thepresent invention be limited to any particular subject. Indeed, avariety of subjects are contemplated. In one embodiment, the subject isa mammal. In a further embodiment, the subject is a mammal selected fromthe group of a human, horse, non-human primate, dog, and cat. In apreferred embodiment, the subject is a human. In an additionalembodiment, the subject is on a low calcium diet.

In one embodiment, the subject is suffering from symptoms ofinflammatory bowel disease. In another embodiment, the subject issuffering from ulcerative colitis. In a different embodiment, thesubject is suffering from Crohn's disease. In a preferred embodiment,the administration of a therapeutic composition comprising abiologically active vitamin D compound reduces the symptoms of disease(i.e. reduces the symptoms of inflammatory bowel disease, ulcerativecolitis, or Crohn's disease). In another embodiment, the biologicallyactive vitamin D compounds are administered under conditions such thatthe symptoms of IBD are reduced. In a different embodiment, the subjectis at risk for inflammatory bowel disease, and the therapeuticcomposition is administered prophylactically. In still furtherembodiments, a therapeutically effective amount of a biologically activevitamin D compound is administered to the subject.

It is not intended that the present invention be limited to particularbiologically active vitamin D compounds. A variety of biologicallyactive vitamin D compounds are contemplated. In one embodiment, thebiologically active vitamin D compound is selected from vitamin D, 1,25dihydroxyvitamin D₃, 1α-hydroxyvitamin D₃, 1,25-dihydroxyvitaminD_(2, 1)α-hydroxyvitamin D₂, 1α,25-(OH)₂-16-ene-D₃,1α,25-(OH)₂-24-oxo-16-ene-D₃, 1α,24R(OH)₂-D₃, 1α,25(OH)₂-22-oxa-D₃,20-epi-22-oxa-24a,24b,-dihomo-1α,25(OH)₂-D₃,20-epi-22-oxa-24a,26a,27a,-trihomo-1α,25(OH)₂-D₃,20-epi-22-oxa-24homo-1α,25(OH)₂-D₃,1,25-(OH)₂-16,23E-diene-26-trifluoro-19-nor-D₃. In a preferredembodiment, the biologically active vitamin D compound is selected from1,25-dihydroxyvitamin D₃, 19-nor-1,25-dihydroxyvitamin D₂,19-nor-1,25-dihydroxy-21-epi-vitamin D₃,1,25-dihydroxy-24-homo-22-dehydro-22E-vitamin D₃, and19-nor-1,25-dihydroxy-24-homo-22-dehydro-22E-vitamin D₃, andnonsecosteroidal vitamin D mimics. In a particularly preferredembodiment, the biologically active compound is 1,25-dihydroxyvitaminD₃. In an additional embodiment, the biologically active vitamin Dcompound is selected from the analogs represented by the followingformula:

-   -   wherein X¹ and X¹ are each selected from the group consisting of        hydrogen and acyl;    -   wherein Y¹ and Y² can be H, or one can be O-aryl or O-alkyl        while the other is hydrogen and can have a β or α configuration,        Z¹ and Z² are both H, or Z¹ and Z² taken together are CH₂; and    -   wherein R is an alkyl, hydroxyalkyl or fluoroalkyl group, or R        may represent the following side chain:    -   wherein (a) may have an S or R configuration and wherein R¹        represents hydrogen, hydroxy or O-acyl, R² and R³ are each        selected from the group consisting of alkyl, hydroxyalkyl and        fluoroalkyl, or, when taken together represent the group        —(CH₂)m— where m is an integer having a value of from 2 to 5, R⁴        is selected from the group consisting of hydrogen, hydroxy,        fluorine, O-acyl, alkyl, hydroxyalkyl and fluoroalkyl, R⁵ is        selected from the group consisting of hydrogen, hydroxy,        fluorine, alkyl, hydroxyalkyl and fluoroalkyl, or, R⁴ and R⁵        taken together represent double-bonded oxygen, R⁶ and R⁷ taken        together form a carbon-carbon double bond and R⁸ may be H or        CH₃, and wherein n is an integer having a value of from 1 to 5,        and wherein the carbon at any one of positions 20, 22, or 23 in        the side chain may be replaced by an O, S, or N atom.

In one embodiment, the biologically active vitamin D compounds of thepresent invention do not cause symptoms of hypercalcemia whenadministered to a subject. In another preferred embodiment of thepresent invention, the biologically active vitamin D compounds of thepresent invention do not generate as much (i.e. a lesser degree) of acalcemic response as compared to calcitriol when administered to asubject. In one embodiment, the biologically active vitamin D compoundshave low calcemic response characteristics, inhibit cell proliferationand promote cell differentiation as compared to calcitriol. In anotherembodiment, these compounds are selected from 1α,25-(OH)₂-24-epi-D₂,α,25-(OH)₂-24a-Homo-D₃, 1α,25-(OH)₂-24a-Dihomo-D₃,1α,25-(OH)₂-19-nor-D₃, and 20-epi-24-homo-1α,25-(OH)₂-D₃.

The present invention also contemplates the administration of atherapeutic composition comprising more than one of the biologicallyactive compounds of the present invention. In other embodiments, thebiologically active compounds of the present invention are administeredin therapeutically effective amounts. In one embodiment, a preferreddose of the biologically active vitamin D compound for the presentinvention is the maximum that a patient can tolerate and not developserious hypercalcemia. In another embodiment, if the biologically activevitamin D compound is not a 1α-hydroxy compound, a daily dose between1.0 and 100 μg per day per 160 pound patient is administered, while aparticularly advantageous daily dose is between 5.0 and 50 μg per dayper 160 pound patient. In a different embodiment, if the biologicallyactive vitamin D compound is a 1α-hydroxy compound, a daily dose ofbetween 0.1 and 20 μg per day per 160 pound patient is administered,while a preferred dose is between 0.5 and 10 μg per day per 160 poundpatient. In a particularly preferred embodiment, the dose is between3-10 μg per day. In an additional embodiment, the therapeuticadministration of the biologically active vitamin D compounds does notcause serious hypercalcemia. In another embodiment, the therapeuticadministration of the biologically active vitamin D compounds onlycauses mild hypercalcemia. In another embodiment, the biologicallyactive vitamin D compounds do not cause symptoms of hypercalcemia.

It is not intended that the present invention be limited to a particularmode of administration. A variety of modes of administration arecontemplated, including intravenously, intramuscularly, subcutaneously,intradermally, intraperitoneally, intrapleurally, intrathecally, orally,rectally and topically. In certain embodiments, the therapeuticcompositions are administered via suppository, or in tablet or capsuleformulations for oral delivery. In one embodiment, administration of thetherapeutic compositions occurs at night. In another embodiment,multiple doses (e.g. 3 or 4) are provided in a 24 hour period. In afurther embodiment, the administration of the therapeutic composition isby pulse intravenous therapy. In a particularly preferred embodiment,the therapeutic compositions are administered via a transdermal patch(skin patch).

The present invention also provides a method of treatment, comprising,providing a subject with symptoms of inflammatory bowel disease and atherapeutic composition comprising a biologically active vitamin Dcompound, and administering the therapeutic compound to the subject. Inone embodiment, the biologically active vitamin D compounds areadministered to a patient after the surgical removal of damaged tissue.In a preferred embodiment, the present invention provides a method oftreatment, comprising, providing a human patient with symptoms ofinflammatory bowel disease, a therapeutic composition comprising abiologically active vitamin D compound, and administering thetherapeutic composition to the patient under conditions such that saidsymptoms are reduced.

The present invention also provides a method of treatment, comprising,providing a subject at risk for inflammatory bowel disease and atherapeutic composition comprising a biologically active vitamin Dcompound, and prophylactically administering the therapeutic compound tothe subject. In a preferred embodiment, the prophylactic administrationof the biologically active vitamin D compounds delays the onset of thesymptoms of inflammatory bowel disease. In a particularly preferredembodiment, the prophylactic administration of the biologically activevitamin D compounds prevents the onset of one or more symptoms ofinflammatory bowel disease (e.g. prevents the onset of abdominal pain,diarrhea, rectal bleeding, weight loss, fever, loss of appetite,dehydration, anemia, or malnutrition, or any combination thereof).

The present invention also provides a composition of matter comprising atransdermal patch, wherein said transdermal patch comprises atherapeutic composition comprising biologically active vitamin Dcompounds. In one embodiment, the transdermal patch comprises atherapeutically effective amount of a biologically active vitamin Dcompound. In another embodiment, the transdermal patch further comprisesa single polymer. In an additional embodiment, the transdermal patchfurther comprises multiple polymers. In another embodiment, thetransdermal patch further comprises a polyurethane acrylic copolymer. Inanother embodiment, the transdermal patch further comprises silicone orpolyisobutylene or both. In a preferred embodiment, the transdermalpatch is worn by a subject at risk for Inflammatory Bowel Disease. Inanother preferred embodiment, the transdermal patch is worn by a subjectwith symptoms of Inflammatory Bowel Disease. In another embodiment, thetransdermal patch delivers biologically active vitamin D compounds to asubject in a continuous manner under conditions such that symptoms ofIBD are reduced.

DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts six biologically active vitamin D compounds useful in thepresent invention.

FIG. 2 depicts five nonsecosteroidal vitamin D compounds (mimics) usefulin the present invention.

DESCRIPTION OF THE INVENTION

The present invention relates to therapeutic compositions and methodsfor the prevention treatment of IBD, and in particular the preventionand treatment of IBD in humans as well as other animals through the useof biologically active vitamin D compounds.

I. Intestinal Bowel Disease Risk Factors and Vitamin D

Intestinal Bowel Disease (IBD) encompasses both Crohn's Disease (CD) andUlcerative Colitis (UC). CD and UC are distinct, but related polygenicdisorders. Familial clustering of IBD strongly suggests that IBDsusceptibility genes contribute to disease development. The relativerisk of IBD among siblings of CD patients was 36.5 and among siblings ofUC patients was 16.6, compared to the general population. Possible IBDsusceptibility genes have been mapped to chromosomes 2, 3, 6, 7, 12 and16. The identity and function of these susceptibility genes is unknown[See, Satangi et al., Clin. Sci. (Colch), May;94(5):473-8 (1998)].

Vitamin D stimulates bone mineralization, and there is evidence for poorbone mineralization in IBD patients [Andreassen et al., Scand. J.Gastroenterol., 32:1247-1255 (1997)]. Many reports found osteoporosis ingreater than 40% of adult CD patients, while pediatric CD patients hadsignificantly decreased bone length and mineral density compared tocontrols [Boot et al., Gut, 42:188-94 (1998)]. Neither malabsorption,nor steroid use explained the poor bone mineralization. Recently, an IBDsusceptibility locus was mapped to chromosome 12 [Satangi et al., Clin.Sci. (Colch), May;94(5):473-8 (1998), and Duerr et al., Am. J. Hu. Gen.,63:95-100 (1998)]. This IBD susceptibility locus is in the same regionas the vitamin D receptor and the 25-hydroxyvitamin D₃-1-α-hydroxylasegenes [Labuda et al., J. Bone Min. Res. 7:1447-53 (1992)].

It is not intended that the present invention be limited to a particularmechanism of action. Indeed, an understanding of the mechanism is notnecessary to make and use the present invention. However, it is believedthat there may be a genetic defect in calcitriol synthesis, transport,or utilization which underlies a dual phenotype of decreased bonemineralization and susceptibility to IBD in some individuals. As such,the therapeutic affect of the administration of biologically activevitamin D compounds to patients may be achieved (at least in part) bycompensating for these genetic defects.

Inheriting IBD susceptibility genes, however, is not sufficient fordisease development. Environmental risk factors may be of equal orgreater significance in determining IBD. IBD prevalence studies show astriking latitude gradient in the United States [Sonnenber et al.,Gastroenterol. 102:1827 (1992)], and Europe [Shivananda et al., Gut,39:690-7 (1996)]. The IBD incidence is high at northern latitudes andlow at southern latitudes. For Example, there are 21 UC patients per100,000 population aged 25-34 years in Norway [Moum et al., Scand. J.Gastroenterol., 31:362-6 (1996)], but only 1 UC patient per 100,000population in Oman [Radhakrishnan et al., Digestion, 58:266-70 (1997)].Therefore, while the IBD-determining environmental risk factor has notbeen identified yet, it appears to correlate strongly with latitude.

Vertebrates, including humans, obtain the majority of the dailyrequirement of vitamin D from casual exposure to sunlight [Holick, M.F., Am. J. Clin. Nutr., Mar;61(3 Suppl.):638S-645S (1995)]. UV lightfrom the sun (282-310 nm) catalyzes a chemical photolysis reaction inthe skin, converting 7-dehydrocholesterol to previtamin D₃, whichspontaneously isomerizes to vitamin D₃ [Holick, M. F., Bone, 7:66-69(1990)]. Vitamin D₃ is biologically inert, and must be activated by25-hydroxylation in the liver and 1α-hydroxylation in the kidney toproduce the vitamin D hormone 1α,25-dihyroxyvitamin D₃ (calcitriol)[Haussler et al., J. Endocrinol. 154:557-573 (1997)]. Above 52°N, thereis insufficient sunlight intensity to catalyze vitamin D biosynthesisfrom October to April, whereas vitamin D biosynthesis occurs year-roundbelow 34°N [Holick, M. F., Am. J. Clin. Nutr., March;61(3Suppl.):638S-645S (1995)]. Vitamin D deficiency is common at northernlatitudes. In one study, at 42°N, vitamin D deficiency affected 57% of arandom population sample [Thomas et al., N. Eng. J. Med., 338:777-783(1998)].

It is not intended that the present invention be limited to a particularmechanism of action. Indeed, an understanding of the mechanism is notnecessary to make and use the present invention. However, it is believedthat insufficient sunlight for vitamin D₃ biosynthesis may be adisease-determining environmental risk factor for IBD. As such, thetherapeutic affect of the administration of biologically active vitaminD compounds to patients may be achieved by compensating for insufficientvitamin D biosynthesis in certain patients.

There is evidence that immune responses to GI tract microbes areinvolved in IBD pathology, but no specific microbe or microbial antigenhas been implicated in IBD etiology [Fiocchi, C., Gastroenterology,115:182-205 (1998)]. Antibodies and cells reactive with autoantigenshave been detected, but their relevance to disease etiology is debated.T lymphocyte infiltration of the mucosa is characteristic of IBD; theseinfiltrating cells display activation markers [Pallone et al., Gut,28:745-753 (1987], and broad specificity [Kaulfersch et al.,Gastroenterology, 95:364-370 (1988)]. In CD, mucosal T cells displayupregulated IL-2 receptors, and spontaneously produce IL-2 and IFN-γ[Breese et al., Immunology, 78:127-31 (1993)]. Moreover, CD mucosalcells spontaneously secrete IL-12 [Monteleone et al., Gastroenterol.,112:1169-78 (1997)]. These observations, together with the consistentfinding of upregulated proinflammatory cytokines in IBD mucosa [Fiocchi,C., Gastroenterology, 115:182-205 (1998)], has suggested the hypothesisthat IBD represents a dysregulated, T helper type 1 (Th1) driven immuneresponse to the constant antigenic stimulation in the gut, with tissuedamage being attributable to inflammatory mediators [Powrie, F.,Immunity, 3:171-74 (1995)]. The opposing anti-inflammatory responsesthat maintain tolerance to enteric antigens appear to be dysfunctionalin IBD.

Knockout animal models in the cytokine genes IL-2 [Sadlack et al., Cell,75:253-61 (1993)], IL-10 [Kuhn et al. Cell, 75:63-74 (1993)], and TGF-β1[Shull et al., Nature, 359:693 (1992)] all spontaneously develop achronic IBD that shares histopathological features with human IBD. The Tcell receptor (TCR) α mutant, TCR β mutant, and class II majorhistocompatibility complex (MHC) mutant mice also develop spontaneousIBD [Mombaerts et al., Cell. Oct. 22;75(2):274-82 (1993)]. In these andother animal models of IBD, Th1 cells have been implicated in diseasepathology [Berg et al., J. Clin. Invest. 98:1010-20 (1996)], while cellsproducing TGF-β have a particularly important regulatory role assuppressors of unchecked, Th1-driven inflammatory colitis [Powrie etal., J. Exp. Med. 183:2669-74 (1996)].

It has been shown that calcitriol is a potent and selective inhibitor ofautoimmune disease in mice. For example, calcitriol administered to miceblocked disease induction for EAE [Cantorna et al., Proc. Natl. Acad.Sci. USA, 93:7861-7864 (1996)], Lyme arthritis and collagen inducedarthritis [Cantoma et al., J. Nutr. 128:68-72 (1998)]. Furthermore,administering calcitriol to animals before EAE induction, or to animalswith stage 1 EAE, arrested the clonal expansion of Th1 cells producingIFN-γ and TNF-α, and stimulated an increase in transcript accumulationfor anti-inflammatory cytokines TGF-β1 and IL-4, in the central nervoussystem and lymph nodes [Cantorna et al., J. Immunol. 160:5314-5319(1998)]. It has been shown that cells producing TGF-β1 have a crucialregulatory role as opponents of unchecked, Th-1 driven inflammatoryresponses [Shull et al., Nature 359:693 (1992), and Powrie et al., J.Exp. Med. 183:2669-74 (1996)].

It is not intended that the present invention be limited to a particularmechanism of action. Indeed, an understanding of the mechanism is notnecessary to make and use the present invention. However, it is believedthat the administration of calcitriol increases TGF-β1 expression, thusstimulating a therapeutic anti-inflammatory response in subjects withinflammatory bowel diseases.

II. Treatment of IBD With Biologically Active Vitamin D Compounds

The present invention contemplates the prevention and treatment ofInflammatory Bowel Disease in a subject through the use of biologicallyactive vitamin D compounds. Various forms of treatment and dosages arecontemplated, as well as the avoidance of the development of thesymptoms of hypercalcemia.

A. Treatment and Dosage

The present invention contemplates both the treatment and prevention ofIBD in humans as well as other animals (e.g. mammals) with biologicallyactive vitamin D compounds in therapeutic amounts. It is not intendedthat the present invention be limited to particular dosages. A broadrange of dosages for the therapeutic administration of the biologicallyactive vitamin D compounds are contemplated. In one embodiment, apreferred dose of the biologically active vitamin D compound for thepresent invention is the maximum that a patient can tolerate and notdevelop serious hypercalcemia. If the biologically active vitamin Dcompound is not a 1a-hydroxy compound, a daily dose between 1.0 and 100μg per day per 160 pound patient is administered, while a particularlyadvantageous daily dose is between 5.0 and 50 μg per day per 160 poundpatient. If the biologically active vitamin D compound is a 1α-hydroxycompound, a daily dose of between 0.1 and 20 μg per day per 160 patientis administered, while a preferred dose is between 0.5 and 10 μg per dayper 160 pound patient. In a particularly preferred embodiment, the doseis between 3-10 μg per day. In general, a preferred dose is the highestamount of the biologically active vitamin D compound the patient cantolerate. The dose is preferably divided between two, three, four orfive treatments within a 24 hour period.

In the United States, the accepted safe dose of 1,25(OH₂)D₃ and19-nor-21-epi-1,25(OH₂)D₃ in patients having normal calcium intakes isbetween 0.5 and 15 μg per day for 1,25(OH₂)D₃, and is 10-20 μg/day for19-nor-1,25-(OH)₂D₂. Therefore, a preferred dose for patients withnormal calcium intakes is between 0.5 and 0.75 μg per day for a 160pound patient depending on the compound administered. Patients on a lowcalcium diet, and/or if the compounds are administered at night, maytolerate more per day (e.g., 3 μg more per day). Therefore, in oneembodiment of the present invention, treatment with biologically activevitamin D compounds is administered in as high a dose as the patient cantolerate without developing symptoms of hypercalcemia. In this regard,high doses (e.g. 3-10 μg per day) are administered. However, for19-nor-1,25(OH₂)D₂ and 24-homo-22-dehydro-22E-1α,25(OH₂)D₃ the safedosage range is 10-20 μg per day per 160 pound patient.

A determination of whether a patient is benefiting from treatment (i.e.wherein symptoms are reduced), is performed by monitoring thequalitative and quantitative symptoms of IBD. Qualitative symptoms whichmay monitored include, but are not limited to, abdominal pain, diarrhea,rectal bleeding, weight loss, fever, loss of appetite, dehydration,anemia, and malnutrition. Quantitative symptoms which may be monitoredinclude, but are not limited to, weight loss, fever, and anemia (using ablood test). A successful treatment is indicated wherein the symptoms ofIBD are reduced. Preferably, treatment should continue as long as IBDsymptoms are suspected or observed.

A determination of whether a subject would benefit from prophylactictreatment of IBD is determined by assessing various risk factors. Inother words, a determination of whether a subject is at risk for IBD ismade. It is not intended that the present invention be limited toparticular risk factors. Indeed, a variety of risk factors may bemonitored, including, but not limited to; genetic predisposition, amountof sunlight the patient normally receives, the age of the patient(common in young people), nationality (common in U.S, England, andNorthern Europe), and ancestry/race (common in people of Jewish decent).Patients at risk are prophylactically administered the therapeuticcompositions of the present invention to delay or prevent the onset ofsymptoms of IBD.

B. Hypercalcemia

As mentioned above, a preferred dose of vitamin D compound for thepresent invention is the maximum that a patient can tolerate and notdevelop serious hypercalcemia. Hypercalcemia is a risk in theadministration of biologically active vitamin D compounds (e.g.calcitriol) because the major physiological function of vitamin D is tomaintain extracellular calcium levels within a very limited normal rangefor normal cellular and metabolic processes (including neuromuscularfunction and bone mineralization). To maintain serum calcium levels,calcitriol primarily increases intestinal absorption of dietary calciumand phosphate, and when required, mobilizes bone calcium. Thus,calcitriol has a potent calcemic effect (i.e. generates a calcemicresponse in a subject). Therefore, the primary concern associated withadministering calcitriol or its analogues to subjects (e.g. humans orother mammals) is elevated serum calcium (hypercalcemia) and phosphatelevels, a condition accompanied by a corresponding increase in urinarycalcium excretion (hypercalcuria).

The toxicity of vitamin D compounds can have serious consequences forrenal function; prolonged hypercalcemia can result in calcium depositionin the kidneys (nephrocalcinosis), kidney stones (nephrolithiasis), andultimately in renal dysfunction leading to uremia. Vitamin Dintoxication may also have serious consequences for neurologicalfunctions. In severe hypercalcemia, the threshold for excitation ofnerve and muscles is increased, resulting in clinical manifestations ofmuscle weakness, lethargy, and even coma. Gastrointestinalmanifestations of vitamin D intoxication include constipation, anorexia,nausea, and vomiting, with subsequent fluid loss which exacerbates thehypercalcemic crisis. Hypercalcemia can also affect cardiovascularfunctioning, including shortening of the QT interval, hypotension, andarrhythmias. Therefore, it is important to monitor the development ofhypercalcemia in patients receiving biologically active vitamin Dcompounds. Hypercalcemia may be monitored in a patient by measuring theterminal serum calcium levels.

One way hypercalcemia risks can be minimized in the treatment ofsubjects with biologically active vitamin D compounds is by performingdose-response studies in an animal model of IBD (e.g. DS-induced mice,carageenan-induced guinea pigs, or IL-2, TGF-β1, TCR, MHC, or IL-10knockout mice) employing a chosen biologically active vitamin Dcompound. In certain embodiments, these studies involve assaying thelevel of the biologically active vitamin D compound in the serum andcorrelating dietary dose of this compound to biologically efficacy andthe symptoms of hypercalcemia such that the minimum effective dose isdetermined. Another method of minimizing the risks of hypercalcemiainvolves administering the biologically active vitamin D compound usingtimed drug release methods (e.g. suppository or transdermal patch) or“slow release” biologically active vitamin D derivatives (See, U.S. Pat.No. 5,952,317, hereby incorporated by reference).

While it is not intended that the present invention be limited to aparticular mechanism of action, and indeed, an understanding of themechanism is not necessary to make and use the present invention, it isbelieved that the use of a transdermal patch reduces the risk ofhypercalcemia (caused by mobilization of calcium across the intestinalwall) by preventing a delivery ‘spike’ of the biologically activevitamin D compound. A transdermal patch is believed to deliver acontinuous, lower dosage stream of the biologically active vitamin Dcompounds such that a spike (total dosage all at once) which could causea severe increase in the mobilization of calcium across the intestinewall, is avoided.

III. Biologically Active Vitamin D Compounds

As defined above, biologically active vitamin D compounds of the presentinvention encompass vitamin D compounds which are biologically active invivo, or are acted upon in a subject (i.e. host) such that the compoundbecomes active in vivo. Examples of such compounds include: vitamin D,1,25(OH₂)D₃ and analogs thereof (e.g. 1α-hydroxyvitamin D₃ (1α-OH-D₃),1,25-dihydroxyvitamin D₂ (1,25-(OH)₂D₂), 1α-hydroxyvitamin D₂(1α-OH-D₂), 26,27-hexafluoro-1,25-dihydroxyvitamin D₂ (F₆-1,25-(OH)₂D₃),19-nor-1,25-dihydroxyvitamin D₂ (19-nor-1,25-(OH)₂D₂),1,25-dihydroxy-24(E)-dehydro-24-homo-vitamin D₃ (1,25-(OH)₂-24-homoD₃),19-nor-1,25-dihydroxy-21-epi-vitamin D₃ (19-nor-1,25-(OH)₂-21-epi-D₃),1α,25 dihydroxyvitamin D₃ triacetate and 25-acetyl-1α,25dihydroxyvitamin D₃, 1,25-dihydroxy-24-homo-22-dehydro-22E-vitamin D₃,19-nor-1,25-dihydroxy-24-homo-22-dehydro-22E-vitamin D₃,1α,25-(OH)₂-24-epi-D₂, 1α,25-(OH)₂-24a-Homo-D₃,1α,25-(OH)₂-24a-Dihomo-D₃, 1α,25-(OH)₂-19-nor-D₃, and20-epi-24-homo-1α,25-(OH)₂-D₃). [See, U.S. Pat. Nos. 5,716,946 and5,891,865, and Bouillon et al., Endocr Rev. April;16(2):200-57 (1995),all incorporated herein by reference].

The present invention also contemplates other biologically activevitamin D compounds which may be represented by various functionalclasses. The first functional class are vitamin D compounds whichexhibit significant activity in vivo as inhibitors of autoimmunity (e.g.multiple sclerosis or experimental autoimmune encephalomyelitis, typeone diabetes, arthritis or lyme arthritis or collagen-induced arthritis,glomerulonephritis, thyroidits, systemic lupus erythematosis), and whichexhibit calcemic activity in vivo that is less than or equal to but notmore than calcitriol (i.e. 1,25(OH)₂D₃. Examples of this class include,but are not limited to, 1α,25-dihydroxy-16ene-vitamin D₃ and1α,25-dihydroxy-24-oxo-16ene-vitamin D₃ [See, Lemire et al.,Endocrinology, 135:2818-2821, (1994)]; 1α,24R-dihydroxy-vitamin D₃ [See,Koizumi et al., Int. Arch. Allergy Appl. Immunol., 77:396-404 (1985);1α,25-dihydroxy-22-oxa-vitamin D₃ [See, Abe et al., Endocrinology,124:2645-2647 (1989)];20-Epi-22-oxa-24a,26a,27a-trihomo-1α,25-dihydroxy-vitamin D₃ [Lilleranget al., Clin. Exp. Immunol., 88:301-306 (1992)]; and19-nor-1,25-dihydroxy-vitamin D₃ [See, U.S. Pat. No. 5,716,946].

The second functional class are vitamin D compounds which exhibitsignificant activity in vivo as an inhibitor of transplanted cells,tissue, or organ rejection (e.g. skin graft, heart graft, islet graft,etc.), and exhibit calcemic activity in vivo that is less than or equalto calcitriol. Examples of this class include, but are not limited to,1,25-dihydroxy-16ene-vitamin D₃ [See, Lemire et al., Transplantation,54:762-763 (1992)]; and20-Epi-22-oxa-24a,26a,27a-trihomo-1,25-dihydroxy-vitamin D₃ [See, Veyronet al., Transplant Immunol., 1:72-76 (1993)].

The third functional class are vitamin D compounds which exhibitsignificant activity in an in vitro cell differentiation assay (e.g.HL-60, U-937, NB4, etc.) and exhibit in vivo calcemic activity that isless than or equal to calcitriol. Examples of this class include, butare not limited to1,25-dihydroxy-16,23E-diene-26-trifluoro-19-nor-cholecalciferol [See,Asou et al., Blood, 92:2441-2449 (1998)];11α-vinyl-1α,25-dihydroxy-vitamin D₃ [See, Bouillon et al., J. Biol.Chem., 267:3044-3051 (1992)]; 1α,25-dihydroxy-16ene-23yne-vitamin D₃[Norman et al., Cancer Res., 50:6857-6864 (1990)];24-homo-22-dehydro-22E-1α,25-dihydroxy-vitamin D₃ and1,25-dihydroxy-22ene-24-homo-vitamin D₃ [Perlman et al., Biochemistry,29:190-196).

The fourth functional class are vitamin D compounds (mimics) whichexhibit significant activity as an activator of the nuclear vitamin Dreceptor in an in vitro transcriptional activation assay, while alsoexhibiting a binding affinity for the serum vitamin D binding proteinthat is less than or equal to calcitriol. An example of this classincludes, but is not limited to LG190090, LG190119, LG190155, LG190176,and LG1900178 [See, Boehm et al., Chemistry & Biology 6:265-275 (1999)].

Other biologically active vitamin D compounds are contemplated for usein the present invention, including, but not limited to, compoundsdescribed in: U.S. Pat. No. 5,936,105 (incorporated herein byreference), U.S. Pat. No. 5,932,565 (incorporated herein by reference),U.S. Pat. No. 5,929,056 (incorporated herein by reference), U.S. Pat.No. 5,905,074 (incorporated herein by reference), U.S. Pat. No.5,902,806 (incorporated herein by reference), U.S. Pat. No. 5,883,271(incorporated herein by reference), U.S. Pat. No. 5,877,168(incorporated herein by reference), U.S. Pat. No. 5,872,140(incorporated herein by reference), U.S. Pat. No. 5,811,562(incorporated herein by reference), U.S. Pat. No. 5,786,347(incorporated herein by reference), U.S. Pat. No. 5,756,733(incorporated herein by reference), U.S. Pat. No. 5,716,945(incorporated herein by reference), and U.S. Pat. No. 5,710,142 (hereinincorporated by reference). Other biologically active compounds usefulin the practice of the present invention are indicated by the followingformulas:

-   -   wherein X¹ and X²are each selected from the group consisting of        hydrogen and acyl;    -   wherein Y¹ and Y² can be H, or one can be O-aryl or O-alkyl        while the other is hydrogen and can have a β or α configuration;        Z¹ and Z² are both H, or Z¹ and Z² taken together are CH₂; and    -   wherein R is an alkyl, hydroxyalkyl or fluoroalkyl group, or R        may represent the following side chain:    -   wherein (a) may have an S or R configuration and wherein R¹        represents hydrogen, hydroxy or O-acyl, R² and R³ are each        selected from the group consisting of alkyl, hydroxyalkyl and        fluoroalkyl, or, when taken together represent the group        —(CH₂)_(m)— where m is an integer having a value of from 2 to 5,        R⁴ is selected from the group consisting of hydrogen, hydroxy,        fluorine, O-acyl, alkyl, hydroxyalkyl and fluoroalkyl, R⁵ is        selected from the group consisting of hydrogen, hydroxy,        fluorine, alkyl, hydroxyalkyl and fluoroalkyl, or, R⁴ and R⁵        taken together represent double-bonded oxygen, R⁶ and R⁷ taken        together form a carbon-carbon double bond and R⁸ may be H or        CH₃, and wherein n is an integer having a value of from 1 to 5,        and wherein the carbon at any one of positions 20, 22, or 23 in        the side chain may be replaced by an O, S, or N atom. As used        herein, the term “alkyl” signifies an alkyl radical of 1 to 5        carbons in all isomeric forms, such as methyl, ethyl, propyl,        isopropyl, butyl, isobutyl, pentyl, etc., and the terms        “hydroxyalkyl” and “fluoroalkyl” refer to such an alkyl radical        substituted by one or more hydroxy or fluoro groups        respectively. The term “acyl” means an aliphatic acyl group of 1        to 5 carbons, such as formyl, acetyl, propionyl, etc. or an        aromatic acyl group such as benzoyl, nitrobenzoyl or        halobenzoyl. The term “aryl” signifies a phenyl-, or an alkyl-,        nitro- or halo-substituted phenyl group.

The present invention also contemplates “slow release” derivatives ofthe biologically active vitamin D compounds of the present invention(See U.S. Pat. No. 5,952,317, incorporated herein by reference). Thefollowing formula describes these various derivatives:

-   -   where R⁵ and R⁶ each represent hydrogen, or taken together R⁵        and R⁶ represent a methylene group.

The side chain group R in the above-shown structure represents a steroidside chain of the structure below:

-   -   where the stereochemical center (corresponding to C-20 in        steroid numbering) may have the R or S configuration, (i.e.,        either the natural configuration about carbon 20 or the opposite        unnatural configuration), and where Z is selected from Y, —OY,        —CH₂ OY, —C≡CY and —CH═CHY, where the double bond may have the        cis or trans geometry, and where Y is selected from a radical of        the structure:    -   where m and n, independently, represent the integers from 0 to        5, where R¹ is selected from hydrogen, OX⁴, fluoro,        trifluoromethyl, and C₁₋₅-alkyl, which may be straight chain or        branched and, optionally, bear a hydroxy substituent, and where        R² is selected from hydrogen, fluoro, trifluoromethyl and C₁₋₅        alkyl, which may be straight-chain or branched, and optionally,        bear a hydroxy substituent, and where R³ and R⁴, independently        represent trifluoromethyl or C₁₋₅alkyl, which may be straight        chain or branched and, optionally, bear a hydroxy substituent,        and where R¹ and R², taken together, represent an oxo group, or        an alkylidene group, ═CR²R², or ═CR²R³, or the group        —(CH₂)_(p)—, where p is an integer from 2 to 5, and where R₃ and        R₄, taken together, represent the group —(CH₂)_(q)—, where q is        an integer from 2 to 5. In the above-shown structures X¹, X² and        X⁴ independently represent hydrogen, an acyl group or a        hydrocarbyloxycarbonyl group, and X³ represents an acyl group or        a hydrocarbyloxycarbonyl group, as previously defined herein.

In order to evaluate whether a given vitamin D analog or slow releasederivative is suitable as a biologically active vitamin D compounduseful for the treatment of IBD, an animal model of IBD (e.g. DS-inducedmice, carageenan-induced guinea pigs, or IL-2, TGF-β1, TCR, MHC, IL-10knockout mice, HLA-B27/β2m transgenic rat, trinitrobenzene sulfonic acidinduced colitis in rodents, or spontaneous IBD in cotton-top tamarincolonies held in temperate climates) may be employed [See also, Bouillonet al., Endocr Rev. April;16(2):200-57 (1995)]. The inhibition of bothinduction and established IBD, as well as risk of hypercalcemia areevaluated. Useful biologically active vitamin D compounds reduce thesymptoms of IBD. Especially useful biologically active vitamin Dcompounds reduce the symptoms of IBD, and do not cause substantialhypercalcemia at therapeutic dosages.

IV. Therapeutic Preparations and Combinations

In some embodiments, the present invention contemplates usingtherapeutic compositions of biologically active vitamin D compounds. Itis not intended that the present invention be limited by the particularnature of the therapeutic composition. For example, such compositionscan be provided together with physiologically tolerable liquids, gels,solid carriers, diluents, adjuvants and excipients (and combinationsthereof).

In addition, biologically active vitamin D compounds may be usedtogether with other therapeutic agents, including, but not limited to,salicylates, steroids, immunosuppressants, antibodies or antibiotics.Particular therapeutic agents which may be used with the biologicallyactive vitamin D compounds of the present invention include, but are notlimited to, the following agents: azobenzene compounds (U.S. Pat. No.4,312,806, incorporated herein by reference), benzyl-substitutedrhodamine derivatives (U.S. Pat. No. 5,216,002, incorporated herein byreference), zinc L-carnosine salts (U.S. Pat. No. 5,238,931,incorporated herein by reference), 3-phenyl-5-carboxypyrazoles andisothiazoles (U.S. Pat. No. 5,294,630, incorporated herein byreference), IL-10 (U.S. Pat. No. 5,368,854, incorporated herein byreference), quinoline leukotriene synthesis inhibitors (U.S. Pat. No.5,391,555, incorporated herein by reference), 2′-halo-2′deoxy adenosine(U.S. Pat. No. 5,506,213, incorporated herein by reference), phenol andbenzamide compounds (U.S. Pat. No. 5,552,439, incorporated herein byreference), tributyrin (U.S. Pat. No. 5,569,680, incorporated herein byreference), certain peptides (U.S. Pat. No. 5,756,449, incorporatedherein by reference), omega-3 polyunsaturated acids (U.S. Pat. No.5,792,795, incorporated herein by reference), VLA-4 blockers (U.S. Pat.No. 5,932,214, incorporated herein by reference), prednisolonemetasulphobenzoate (U.S. Pat. No. 5,834,021, incorporated herein byreference), cytokine restraining agents (U.S. Pat. No. 5,888,969,incorporated herein by reference), and nicotine (U.S. Pat. No.5,889,028, incorporated herein by reference).

The therapeutic compositions of the present invention can beadministered to mammals for veterinary use, such as with domesticanimals and non-human primates, and clinical use in humans in a mannersimilar to other therapeutic agents. In general, the dosage required fortherapeutic efficacy varies according to the type of use and mode ofadministration, as well as the particularized requirements of individualhosts. A preferred dose of the biologically active vitamin D compoundsof the present invention is the maximum that a patient can tolerate andnot develop serious hypercalcemia. The attending medical professional iscapable of determining the therapeutically effective dosage based on thecharacteristics of the subject (e.g., gender, age, weight, amount ofcalcium in diet, etc.).

With respect to the mode of administration, in some embodiments thebiologically active vitamin D compounds (and therapeutic compositionsthereof) are administered intravenously, intra-muscularly,subcutaneously, intradermally, intraperitoneally, intrapleurally,intrathecally, orally, rectally or topically. In some embodiments,formulations for such administrations may comprise an effective amountof the biologically active vitamin D compound in sterile water orphysiological saline. In other embodiments, formulations for suchadministrations may comprise an effective amount of the biologicallyactive vitamin D compound in an organic solvent (e.g. ethanol, vegetableoil, or glycerol).

On the other hand, therapeutic compositions may contain such normallyemployed additives as binders, fillers, carriers, preservatives,stabilizing agents, emulsifiers, buffers and excipients as, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharin, cellulose, magnesium carbonate, and the like. Thesecompositions typically contain 1%-95% of active ingredient, preferably2%-70%.

The biologically active vitamin D compounds of the present invention canalso be mixed with diluents or excipients which are compatible andphysiologically tolerable. Suitable diluents and excipients are, forexample, water, saline, dextrose, glycerol, or the like, andcombinations thereof. In addition, if desired, the compositions maycontain minor amounts of auxiliary substances such as wetting oremulsifying agents, stabilizing or pH buffering agents.

In some embodiments, the therapeutic compositions of the presentinvention are prepared either as liquid solutions or suspensions, assprays, or in solid forms. Oral formulations usually include suchnormally employed additives such as binders, fillers, carriers,preservatives, stabilizing agents, emulsifiers, buffers and excipientsas, for example, pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharin, cellulose, magnesium carbonate,and the like. These compositions take the form of solutions,suspensions, tablets, pills, capsules, sustained release formulations,or powders, and typically contain 1%-95% of active ingredient,preferably 2%-70%. One example of an oral composition useful fordelivering the therapeutic compositions of the present invention isdescribed in U.S. Pat. No. 5,643,602 (incorporated herein by reference).

Additional formulations which are suitable for other modes ofadministration, such as topical administration, include salves,tinctures, creams, lotions, transdermal patches, and suppositories. Forsalves and creams, traditional binders, carriers and excipients mayinclude, for example, polyalkylene glycols or triglycerides. One exampleof a topical delivery method is described in U.S. Pat. No. 5,834,016(incorporated herein by reference). Other liposomal delivery methods mayalso be employed. Examples of this type of delivery of the biologicallyactive vitamin D compounds of the present invention include U.S. Pat.Nos. 5,851,548 and 5,711,964 (both incorporated herein by reference).

In certain particularly preferred embodiments, the therapeuticcomposition comprising biologically active vitamin D compounds isadministered via a transdermal patch. A transdermal patch optimallyincludes a therapeutically effective amount of the biologically activevitamin D compounds of the present invention. While not limited to anymechanism, it is believed that transdermal delivery would provide acontinuous supply of the vitamin D compound, maintaining the vitamin Dreceptor occupancy at a stable, optimal level, to achieve the desiredbiological effect. This is in contrast to other modes of delivery (e.g.oral or intravenous) which could provide a peak hormone concentration(spike) shortly after delivery, which would subsequently decline(leading to cyclical hormone occupancy and peak concentrationsstimulating calcium absorption, bone resorption, or soft tissuecalcification). Transdermal delivery may also bypass delivery of thevitamin D compounds to the intestine, decreasing binding to the vitaminD receptors in the intestinal epithelial cells. This in turn maydecrease stimulation of intestinal calcium absorption, and decrease thecalcemic effect of the vitamin D compound. Transdermal delivery may alsobe preferred because intestinal physiology is disturbed in patients withIBD, which may alter uptake of the vitamin D compound in apatient-specific, non-predictable manner (making dose determinationdifficult for other modes of administration such as oral delivery).Transdermal delivery may be more convenient than other modes of delivery(especially for children), and could increase patient compliance.

One example of a transdermal patch for delivering therapeutics employs apolyurethane acrylic copolymer (U.S. Pat. No. 4,638,043, incorporatedherein by reference). Another example of a transdermal patch employspolymers and vitamin E (U.S. Pat. No. 5,830,505, incorporated herein byreference). A third example of a transdermal patch employs an adhesivematrix of silicone or polyisobutylene or both (U.S. Pat. No. 5,876,746,incorporated herein by reference). Other transdermal patches are knownin the art, and are contemplated as modes for delivering thebiologically active vitamin D compounds of the present invention.

In other preferred embodiments, enteric formulations are employed. Thecovering may comprise an enteric coating or a capsule. The terms“enteric coating” or “enteric film” are used interchangeably and referto a material or compound which is resistant to acid pH (i.e., anacid-resistant compound), such as that found in the stomach. An entericcoating when applied to a solid inhibits the dissolution of the solid inthe stomach.

Standard techniques are known to the art for the encapsulation of solidcompositions. These techniques include microencapsulation of a solidcomposition wherein an enteric coating is applied to the solidcomposition. The coated material may be delivered orally to a subject bysuspending the microencapsulated particles in pharmaceutical suspensionsolutions known to the art. The capsule would preferably have thecharacteristic of being resistant to dissolution in the stomach andbeing capable of dissolving in the intestines. Numerous suitable capsuleformulations are available to the art; in addition standard techniquesare available for the filling of capsules including the use of inertfiller materials to provide sufficient bulk of the filling of a capsulewith a therapeutic composition in a solid form. In addition to the useof encapsulated compositions, the biologically active vitamin Dcompounds may be delivered orally in tablet or pill form. Thebiologically active vitamin D compounds may be combined with inertmaterials to provide sufficient bulk for the pressing of the tablet orpill. Once formed, the tablet or pill may then be coated with an entericfilm to prevent dissolution in the stomach and to enhance dissolution inthe intestines.

Experimental

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: N (normal); M (molar); mM (millimolar); μM(micromolar); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); pmol (picomoles); g (grams); mg (milligrams); ug(micrograms); ng (nanograms); l or L (liters); ml (milliliters); μl(microliters); cm (centimeters); mm (millimeters); μm (micrometers); nm(nanometers); DS (dextran sulfate); ° C. (degrees Centigrade); Sigma(Sigma Chemical Co., St. Louis, Mo.); and

EXAMPLE 1 Calcitriol Inhibition of IBD Induction

This Example describes the inhibition of IBD induction in mice withcalcitriol. Two types of mouse models are utilized. The fist group isthe widely used DS-induced murine model, which reflects the involvementof enteric microbes, innate immunity, and non-specific inflammation inIBD. Mouse strains vary in their susceptibility to DS-induced IBD[Mahler et al., Am. J. Physiol.—Gastro. & Liver Physiol., 37 G: 544-G551, (1998)], so the highly susceptible strains C3H/HeJ and C57BL/6J areemployed. The second group of IBD models involves mice with targeteddisruptions in immunologically relevant genes. In particular, the widelyused IL-10 knockout on the C57BL/6J background is employed (i.e.C57BL/6J-IL-10 ko). An alternative to these C57BL/6J-IL-10 ko mice (notdescribed in this Experiment), are mice with a homozygous IL-10 komutation on the mixed 129/Ola×C57BL/6 genetic background. These twogroups of mice are employed as described below to demonstrate the effectof calcitriol on the inhibition of IBD induction (See Table 1, overviewof Experimental Groups).

TABLE 1 Experimental Groups. Group Strain Inducing agent Treatment 1C3H/HeJ none none 2 C3H/HeJ DS none 3 C3H/HeJ DS calcitriol 4 C3H/HeJ DSprednisolone 5 C57BL/6J none none 6 C57BL/6J DS none 7 C57BL/6J DScalcitriol 8 C57BL/6J DS prednisolone 9 129/O1a × C57B1/6J-IL-10 ko nonenone 10 129/O1a × C57BL/6J-IL-10 ko none calcitriol 11 129/O1a ×C57BL/6J-IL-10 ko none prednisolone

A. Treatment of the Mice

The DS-induced IBD study (groups 1-8; Table 1) are treated as follows.Beginning on day 0 and continuing, groups of 10 mice are fed a purifieddiet without or with calcitriol (50 ng/day females; 200 ng/day males);this dose is based on dose-response studies in EAE [Cantoma et al., J.Immunol., 160:5314-5319 (1998)]. The prednisolone-treated group is fed apurified diet with prednisolone (20 ng/day). These experimental dietshave been published [Cantoma et al., Proc. Natl. Acad. Sci. USA,97:7861-7864 (1996)], and are replenished each 2-3 days. On day 2, themice are weighed and DS (3.5% wt/vol) is given in the drinking water ondays 2-6 (Okayasu, et al., Gastro., 98:694-702 (1990)]. The mice receiveacidified drinking water without DS for days 7-22. On days 7, 11, 15,and 19, mice are weighed and stool samples are collected. A blood sampleis collected on day 11. On day 22, mice are weighed, euthanized, andstool, blood, and colon samples are collected. The samples are analyzedas described below.

The mock-treated control mice in this Example are expected to showsevere weight loss, bloody diarrhea (as evidenced by fecal hemoglobin),shortening of the colon and thickening of the colonic wall, mucosalulceration, goblet cell loss, and crypt elongation and loss (asevidenced by histopathologic score), and inflammatory infiltration bylymphocytes, macrophages, neutrophils, and granulocytes (as evidenced byfecal lactoferrin and colonic myeloperoxidase activity) during IBDinduction and progression. The calcitriol-treated mice exhibitsignificantly reduced incidence or severity of disease as compared tothe mock-treated controls. With respect to severity, thecalcitriol-treated mice exhibit significantly reduced weight loss,bloody diarrhea, shortening and thickening of the colon, histopathologicscore, and inflammatory infiltration as compared to the mock-treatedcontrols.

The spontaneous IBD study (groups 9-11; Table 1) are treated the same asthe DS-induced mice, except purified diet feeding begins when theIL-10-ko mice are weaned at age 3 weeks, and no DS is administered. Miceare weighed and stool samples are collected twice weekly for four weeks.A blood sample is collected at age 5 weeks. After four weeks, mice areweighed, euthanized, and stool, blood, and colon samples are collected.These samples are analyzed as described below in Example 2.

The mock-treated control mice carrying a null-mutation of the IL-10 genedescribed in this Example are expected to show growth retardation by age4 weeks (as evidenced by low blood hemoglobin levels), and a significantmortality incidence by 10 weeks. These mice are also expected to showclinical signs of IBD, specifically bloody fecal lactoferrin and colonicmyeloperoxidase activity, perianal ulceration, and occasional rectalprolapse. Finally, these mice are expected to show histopathologicalsigns of IBD, specifically inflammatory infiltration with lymphocytes,plasma cells, marcophages, and neutrophils, ulceration, abnormalenlarged and branched crypts, branched and fused villus structures, andmarked hyperplasia of the mucosa leading to thickening of the intestinalwall. The calcitriol-treated mice exhibit significantly reduced growthretardation, anemia, mortality, bloody diarrhea, intestinalinflammation, perianal ulceration and rectal prolapse, andhistopathologic score as compared to the mock-treated controls.

EXAMPLE 2 Analysis of Samples from Treated Mice

This Example describes the analysis of stool, serum, and colon samplesfrom both groups of mice described in Example 1. Stool extracts areanalyzed individually for protein, hemoglobin, and lactoferrin. Serumsamples are analyzed for Ca, IFN-γ, and TGF-β1. Colon samples areanalyzed individually for myeloperoxidase (5/group), IFN-γ and TGF-β1immunohistochemistry, and for histopathology (5/group). Fecal extractsand intestinal tissue extracts are assayed for protein content by amicro-Bradford assay [Bradford, M. M., Anal. Biochem., 72:248-254(1976)]. Results of the hemoglobin, lactoferrin, and myeloperoxidaseassays are then determined per mg of protein in the sample. Fecalhemoglobin and fecal lactoferrin (as well as weight) are plotted as afunction of time. Histopathology scores for disease incidence andseverity, myeloperoxidase activity, and IFN-γ and TGF-β1 values aretabulated, and differences between groups are compared by thenonparametric Wilcoxon test (Schefler, W. C., Statistics for theBiological Sciences, 2nd Edition, Addison-Wesley Pub. Co., Reading,Mass., USA, 1979).

A. Stool Sample Assays

Intestinal bleeding in a breached intestinal barrier is measured in liveanimals by measuring fecal hemoglobin levels, which have been shown tobe elevated in UC patients [Saitoh, et al., Digestion 56:67-75 (1995)].Stool samples, collected over a 24 hour period are weighed, homogenizedin a small amount of water, and centrifuged in order to detect therelative hemoglobin concentration of each sample (ng/mg of protein).

The fecal hemoglobin two-site ELISA is performed on these samples asdescribed [Saitoh, et al., Digestion, 56:67-75 (1995)] withmodifications. All ELISA steps are separated by washing four times(TRIS-buffered saline with 0.05% Tween 20). ELISA plates (Immulon,Dynatech; 96-well) are coated overnight in the cold with rabbitpolyclonal antibodies (pAb) to mouse hemoglobin (ICN Biomedical ResearchProducts; 5 μg/ml in 0.1 M sodium carbonate buffer pH 8.2), and blockedwith 0.1 M TRIS-buffered saline (pH 8) containing 1% bovine serumalbumin. The samples and the hemoglobin standard (Sigma) are seriallydiluted in buffer (0.1 M TRIS-buffered saline, pH 7.5, with 0.1% bovineserum albumin) and aliquots are incubated 1 hour at 37° C. in duplicateassay wells; assay blank wells receive buffer only. An optimal amount ofbiotinylated rabbit pAb to mouse hemoglobin (ICN) is added to each welland incubated 1 hour at 37° C. The assay is developed withtetramethylbenzidine (TMB) plus hydrogen peroxide substrate solution(ICN), the reaction is stopped by adding 100 μl of 1M phosphoric acid,and the color is measured on an ELISA plate reader. The hemoglobin ineach sample is determined from the linear portion of a log-linear plotof A-450 nm versus hemoglobin standard concentration, and results areexpressed in ng/mg protein. The calcitriol-treated mice exhibit lesshemoglobin protein in stool extracts than the mock-treated control mice.

GI tract inflammation in live animals is indicated by measuring fecallactoferrin concentrations. High levels of fecal lactoferrin have beendemonstrated in patients with colorectal diseases [Sugi et al., Am. J.Gastroenterol. 91:927 (1996)]. The fecal lactoferrin two-site ELISA isperformed in the same manner as the hemoglobin assay above, except thatrabbit pAb to human lactoferrin (ICN Biomedical Research Products)replaces rabbit pAb to mouse hemoglobin as the coating Ab, andbiotinylated rabbit pAb to human lactoferrin replaces rabbit pAb tomouse hemoglobin as the detecting Ab. The lactoferrin in each sample isdetermined from the linear portion of a log-linear plot of A-450 nmversus lactoferrin standard concentration. Results are expressed aslactoferrin ng/mg protein. The calcitriol-treated mice show lesslactoferrin protein in stool extracts than the mock-treated controlmice.

B. Colon Tissue Assays

Large intestine of the euthenized mice are collected, and the cecum isseparated from the colon. Intestinal specimens are gently flushed withFekete's acid-alcohol-formalin fixative. The entire colon, including therectum, is prepared as an intestinal roll [Moolenbeek and Ruitenber, LabAnim. 15:57-59 (1981)]. It is placed on an index card and rolled intoconcentric centrifugal circles (in the plane of the card) around acentral toothpick. Intestinal rolls are fixed overnight in Fekete'ssolution, and then transferred to 70% ethanol. The fixed intestinalrolls are then embedded in paraffin, sectioned at 5 μm, and stained withhematoxylin and eosin.

Two intestinal role sections per animal are coded and evaluated by aveterinary pathologist and a researcher without access to the code. Apublished evaluation method is used [Mahler et al., Am. J.Physiol.—Gastro. & Liver Physiol., 37 G: 544-G 551, (1998)]. Theevaluation is based on severity of lesions (graded 0 to 3), andestimated area involved (graded 0 to 4). The severity, ulceration,hyperplasia, and affected area scores are summed and tabulated, with thecalcitriol-treated mice exhibiting a lower histopathological score thanthe mock-treated mice.

Myeloperoxidase assays have been used as objective and quantitativemeasures of inflammation in humans [e.g. Dwarakanath et al., Clin. Sci.92:307-13 (1997)] and animals [e.g. Hogaboam et al., J. Clin. Invest.,100:2766-76 (1997)] with IBD. Accordingly, intestinal inflammation ismeasured in the mouse intestinal tissue samples by measuringmyeloperoxidase activity levels. The myeloperoxidase assay is performedas in Krawisz et al., Gastroenterol. 87:1344-50 (1984), as modified bySchneider and Issekutz, J. Immunol. Methods, 198:1-14 (1996). Briefly,washed intestinal tissue samples (3/mouse; 200 mg each) are minced andhomogenized in hexadecyltrimethylammonium bromide (HTAB) buffer (1 ml;0.5% HTAB in 50 mM phosphate, pH 6) to release the enzyme. Thehomogenate is frozen and thawed four times, then centrifuged. Thesupernatant (10 μl) is added to a well of a 96-well plate containing0.29 ml assay mix (TMB plus hydrogen peroxide substrate solution; ICN),and the A-450 nm is measured on an ELISA plate reader as a function oftime. An assay blank is prepared with a heat-inactivated supernatant andsubtracted. A unit of enzyme activity is defined as the amountcatalyzing the oxidation of 1 μmole substrate/min under theseconditions. Results are expressed as myeloperoxidase U/mg protein. Thecalcitriol-treated mice show less myeloperoxidase protein in theintestinal tissue extracts than the mock-treated control mice.

Intestinal tissue is also analyzed for the presence of IFN-γ and TGF-β1(cytokines thought to regulate mucosal inflammation, see below).Intestinal tissue specimens are snap frozen in OCT embedding compound(Miles Laboratories). Longitudinal 10 μm sections are applied to coatedglass slides, air dried, and fixed briefly in acetone. Sections are thenre-hydrated in PBS with 0.05% Tween 20, and washed in this bufferbetween each subsequent reaction step. All reactants are dissolved inPBS-Tween buffer. Sections are reacted 30 minutes with 3% hydrogenperoxide, then 10 minutes with 3% goat serum, then overnight in the coldwith polyclonal rabbit antibodies to IFN-γ and TGF-β1 or control rabbitserum, and finally with biotinylated goat antibodies to rabbit IgG.Color is developed with the Vector Elite ABC kit. The intestinal tissuesections from the calcitriol-treated mice exhibit qualitatively greaterimmunohistochemical staining with polyclonal rabbit antibodies to TGF-β1and qualitatively less immunohistochemical staining with the polyclonalrabbit antibodies to IFN-γ than the intestinal tissue sections from themock-treated control mice.

C. Blood and Serum Sample Assays

Anemia can be a sign of IBD due to blood loss in the stool. To determinewhether anemia is present, 0.5 ml of blood is obtained on days 11 and 22(prevention of DS-induced mouse IBD models) and at age 5 and 7 weeks(prevention of spontaneous mouse IBD model). A blood hemoglobindetermination is performed. A small blood aliquot is dispensed intolysis buffer [3 mM K₃(FeCN)₆, 1.5 mM KCN, 5 mM Na₂BO₄, 0.1% NonidetP-40] and the absorbance at 546 nm is measured using lysis buffer as ablank. This absorbance is compared to a reference curve produced usingpurified moused hemoglobin dissolved in lysis buffer. The remainingblood is allowed to clot overnight in the cold, centrifuged, and theserum is collected for further analysis.

As hypercalcemia is a possible serious side-effect of calcitrioladministration [Chan et al., Cur. Prob. Surgery, 34:445-523 (1997)],calcitriol-treated and control mice are monitored for terminal serumcalcium. Serum calcium is measured by a colormetric reaction (SigmaDiagnostics). Calcium ions form a purple complex with o-cresolphthaleincomplexone at alkaline pH (0.5 M 2-amino-2-methyl-1,3-propanediolbuffer); 8-hydroxyquinoline (0.25%) is included to prevent magnesium ioninterference. Sample, standard, or buffer blank (10 μl) is added toduplicate tubes of a working solution (1 ml) of equal parts Ca bindingreagent and Ca buffer. After 5 minutes, 0.3 ml of each reaction istransferred into a 96-well plate and the absorbance at 575 nm ismeasured on an ELISA plate reader. Results of this assay aid in thedetermination the proper level and time course of calcitrioladministration to avoid hypercalcemia for subjects treated for IBD withcalcitriol.

In previous studies, administration of calcitriol to mice with EAEarrested the clonal expansion of Th1 cells producing IFN-γ, andstimulated TGF-α1 transcript synthesis in the central nervous system anddraining lymph nodes [Cantorna et al., J. Immunol., 160:5314-5319(1998)]. Reciprocal IFN-γ and TGF-β1 responses are thought to regulatemucosal inflammation [Strober et al., Immunol. Today, Feb;18(2):61-4(1997)], and TGF-β1 responses are crucial to suppress inflammatorycolitis [Powrie et al., J. Exp. Med., 183:2669-74 (1996)]. As such, atwo-step ELISA is performed to analyze IFN-γ (PharMingen) and TGF-β1(Promega Corp.) as immune response markers in the serum of thecalcitriol-treated mice compared to mock-treated mice.

EXAMPLE 3 Treating Established IBD in Mice

This example describes the treatment of established IBD in mice usingcalcitriol. The procedure is the same as Example 1 above, excepttreatment is not started until the mice show signs of IBD. This isaccomplished by administering DS in the drinking water of the micelisted in groups 1-4 in Table 1, followed by acidified water without DSthereafter. The mice are also weighed at the beginning of thisprocedure. At two-day intervals, stool samples are tested as in Example1 for hemoglobin and lactoferrin. Treatment with calcitriol is begunwhen these test indicate the mice are suffering from IBD.

Thereafter, weights and stool samples are obtained at 4-day intervals.On day 22, the mice are weighed, euthanized, and stool, blood, and colonsamples are collected. Stool extracts, blood, serum, and colon samplesare analyzed as described in above in Example 2. Weight, fecal and bloodhemoglobin, and fecal lactoferrin are plotted as a function of time.Disease incidence, severity (as evidenced by histopathologic score),myeloperoxidase activity, and IFN-γ and TGF-β1 values are thentabulated. The results of this experiment indicate that calcitrioltreatment of mice exhibiting symptoms of IBD exhibit reduced symptoms ofdisease compared to controls.

EXAMPLE 4 Therapeutic and Prophylactic Use of Calcitriol to Treat andPrevent IBD

Therapeutic formulations of calcitriol are used prophylactically andtherapeutically to treat IBD in humans. Individuals at risk ofcontracting IBD, particularly young adults, or those or Jewish descentare administered an effective amount of calcitriol in a therapeuticformulation to prevent or reduce the severity of the disease. A patientwith symptoms of IBD is administered an effective dose of calcitrioldaily until symptoms of IBD are reduced.

EXAMPLE 5 Therapetic and Prophylactic Use of Vitamin D to Treat andPrevent IBD

Therapeutic formulations of vitamin D are used prophylactically andtherapeutically to treat IBD in humans. Individuals at risk ofcontracting IBD, particularly young adults, or those or Jewish descentare administered an effective amount of vitamin D in a therapeuticformulation to prevent or reduce the severity of the disease. A patientwith symptoms of IBD is administered an effective dose of vitamin Ddaily until symptoms of IBD are reduced.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith certain preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such embodiments.Indeed, various modifications of the described modes for carrying outthe invention which are obvious to those skilled in biochemistry,immunology, chemistry, molecular biology, the medical profession orrelated fields are intended to be within the scope of the followingclaims.

1. A method of treating inflammatory bowel disease, comprising:administering a therapeutically effective amount of a therapeuticcomposition comprising 1α-hydroxyvitamin D₂ to a subject suffering fromthe symptoms of inflammatory bowel disease, wherein said inflammatorybowel disease is selected from the group consisting of ulcerativecolitis and Crohn's disease.
 2. The method of claim 1, wherein saidtherapeutically effective amount comprises a daily dose of between 0.1μg and 20 μg per 160 pounds of said subject.
 3. The method of claim 1,wherein said therapeutically effective amount comprises a daily dose ofbetween 0.5 μg and 10 μg per 160 pounds of said subject.
 4. The methodof claim 1, wherein said therapeutically effective amount comprises adaily dose of between 3.0 μg and 10 μg per 160 pounds of said subject.5. The method of claim 1, wherein said administering is conducted in acontinuous manner.
 6. The method of claim 1, wherein said administeringis via a transdermal patch.
 7. The method of claim 1, wherein saidadministering is via a suppository.
 8. The method of claim 1, whereinsaid administering is via a slow release oral formulation.
 9. A methodof treating inflammatory bowel disease, comprising: administering atherapeutically effective amount of a therapeutic composition comprising19-nor-1,25-dihydroxyvitamin D₂ to a subject suffering from the symptomsof inflammatory bowel disease, wherein said inflammatory bowel diseaseis selected from the group consisting of ulcerative colitis and Crohn'sdisease.
 10. The method of claim 9, wherein said therapeuticallyeffective amount comprises a daily dose of between 0.1 μg and 20 μg per160 pounds of said subject.
 11. The method of claim 9, wherein saidtherapeutically effective amount comprises a daily dose of between 0.5μg and 10 μg per 160 pounds of said subject.
 12. The method of claim 9,wherein said therapeutically effective amount comprises a daily dose ofbetween 3.0 μg and 10 μg per 160 pounds of said subject.
 13. The methodof claim 9, wherein said administering is conducted in a continuousmanner.
 14. The method of claim 9, wherein said administering is via atransdermal patch.
 15. The method of claim 9, wherein said administeringis via a suppository.
 16. The method of claim 9, wherein saidadministering is via a slow release oral formulation.